Composition and method for stabilizing of biomolecules

ABSTRACT

The present invention relates in general to the field of biotechnology. The invention particularly relates to a composition and process for stabilizing or preserving biological molecules, as well as devices that comprise correspondingly stabilized or preserved biomolecules.

The present invention relates generally to the field of biotechnology. In particular, the invention relates to a composition and process for stabilizing, or, as the case may be, preserving biological molecules as well as devices that comprise the correspondingly stabilized biomolecules.

The use of proteins and polypeptides in industrial products and processes requires large quantities of these biomolecules, in particular for clinical/diagnostic and pharmaceutical purposes. Whereas the required basic techniques such as isolation and purification of proteins in industrial quantities are mostly established, the most difficult aspect of the use of such biomolecules resides in maintenance for the envisioned application, of the desired native, or, active molecular properties. Particularly, in the storage and transport of biomolecules, losses in activity often have to be taken into account, whereby the success of later applications is put in risk. Commercially obtainable protein preparations therefore for the most part comprise compounds whose presence can minimize the activity loss during storage or transport. Furthermore, storage and transport is mostly carried out under low temperatures, although a freezing of, for example, particular proteins may be undesirable because of molecular changes.

It is known from the literature that particular plants and animals have developed mechanisms to survive in the state of approaching complete dehydration. This state of stress is referred to anhydrobiosis and is observed in organisms that are exposed to dry conditions. During anhydrobiosis, the organism finds itself in a kind of resting state until a rehydration allows normal metabolism to continue. Common characteristic property of these organisms is the synthesis of high concentrations of non-reducing sugars that is induced during anhydrobiotic conditions. The observed accumulation of large quantities of trehalose as a response to dehydration in various organisms leads to a protection of membranes and proteins from damage to their molecular integrity and correlates to a certain tolerance with respect to water removal. One assumes that the sugar replaces, or, as the case may be, functionally substitutes the removed water molecules and is involved in the formation of an intracellular organic glass by which, one assumes, the cell contents are stabilized.

In the state of the art, the use of trehalose in the production of antibody-coated microtiter plates is described in order to stabilize this normally very quickly denatureable protein species (V. K. Nguyen et al., Protection of immunoreactivity of dry immobilized proteins on microtitration plates in ELISA: application for detection of autoantibodies in Myasthenia gravis, J. of Biotechnology, 72, pp. 115 to 125 (1999)). In this case, microtiter plates having antibodies immobilized thereon are covered with a bovine serum albumin (BSA) and trehalose-containing film and are thereafter dried. The immobilized dried antibodies of the ELISA plates made in this manner showed a storage capability of up to thirty days even at increased temperatures (up to 50° C).

An increase of the storage capability, particularly from a commercial viewpoint, at room temperature or even tropical conditions can, however, not be achieved with this technology. Particularly in connection with the preparation of large quantities of immobilized proteins, it is desirable to have a storage capability for a time period of over a year with substantially constant biological activity, or functionality of the biomolecule.

A further disadvantage of the above-described technology lies in the use of bovine serum albumin (BSA), a protein mixture of which it is known that, in the framework of antibody-aided applications, unspecific binding with antibodies results and thereby creates undesired cross reactions through which the entire experimental result is negatively influenced.

It is known, particularly from plant seeds and pollen that, in reaction to water removal, proteins of the LEA class are formed in them. (LEA=‘late embryogenesis abundant’) (J. Ingram and D. Bartels, Annu. Rev. Plant Physiol. Plant Mol. Biol., 47 pp. 377-403 (1996)). The LEA proteins also identified in nematodes, comprise a special, 11 amino acid motif, which presumably forms an amphipathic a-Helix through which the oligomerization of the protein is controlled. The LEA proteins are extremely hydrophilic and resistant to denaturation by heat. Initial experiments with a protein of this class purified from the pollen of Typha latifolia have shown that, in vitro, sucrose glasses can be stabilized by incubation with this protein. It is therefore suspected that non-reducing sugars and representatives of the protein class LEA work together in a synergistic manner in the formation of a stable bioglass in the cytoplasma of anhydrobiotic plants and in seeds and pollen being resistant to desiccation (J. Brown et al, Plant desiccation gene found in a nematode, Nature, 416, p. 38 (2002)).

An object of the present invention is therefore the provision of compositions and processes for stabilizing, or, as the case may be, preserving biomolecules, by which the disadvantages of the state of the art are overcome, and with which the desired biological activity of the molecules can be preserved, even without cooling, over a longer period of time.

The object is solved according to the present invention by the composition according to the main claim.

The composition according to the present invention comprises at least one non-reducing disaccharide, selected from the group consisting of trehalose (D-glucopyranosyl-D-glucopyranose), sucrose (β-D-fructofuranosyl-α-D-glucopyranoside), as well as derivatives thereof, and at least one protein or polypeptide of the LEA class.

According to a preferred embodiment, the composition according to the present invention comprises trehalose as well as at least one protein or polypeptide of the LEA class with an 11 amino acid motif, characterized by the following general formula (SEQ ID NO. 1):

-   (1)-(2)-(3)-(4)-(5)-(6)-(7)-(8)-(9)-(10)-E,     wherein

(1) signifies K or T,

(2) signifies A, G. K, M or T,

(3) signifies R, D, A, E, Q or K,

(4) signifies E, K or S,

(5) signifies T, F, Y or A,

(6) signifies K, R, T or A,

(7) signifies D, E or Q,

(8) signifies S, R, Y or K,

(9) signifies A or T, and

(10) signifies G, A or R.

In a particularly preferred embodiment, the composition comprises at least one protein or polypeptide of the LEA subclass 3 having an amino acid sequence that is coded by a nucleotide sequence as deposited in the GenBank under the accession number AF423069 or S39475.

The composition according to the present invention comprises, as particularly preferred, at least one protein or polypeptide of the LEA subclass 3 with an 11 amino acid motif, selected from the group consisting of: (a) K-T-A-E-F-R-D-S-A-G-E, (SEQ ID NO. 2) (b) K-G-Q-E-F-K-E-R-A-G-E, (SEQ ID NO. 3) (c) K-A-E-E-T-K-Q-R-A-G-E, (SEQ ID NO. 4) (d) K-M-D-E-T-K-Q-R-A-G-E, (SEQ ID NO. 5) (e) K-A-R-K-T-K-D-S-A-A-E, (SEQ ID NO. 6) (f) K-A-K-E-Y-K-D-Y-T-A-E, (SEQ ID NO. 7) (g) K-A-R-E-T-T-E-K-A-R-E, (SEQ ID NO. 8) and (h) T-K-D-S-A-A-E-K-A-R-E. (SEQ ID NO. 9)

According to a preferred embodiment, the composition comprises the components of the non-reducing disaccharide and the protein or polypeptide of the LEA class in respective quantities of from 0.01 to 15, or, as the case may be, 0.00001 to 1 weight percent, in each case with reference to a ready-to-use solution.

For example, the composition is produced by adding 0.1 weight percent of a purified LEA protein and 5 weight percent trehalose to a solution of 50 mM phosphate and 100 mM NaCl with a pH value of 6.8. If desirable, further components such as, for example, sodium azide (0.02 weight percent) can also be added.

It is clear to one of skill in the art that by means of the sequence and motif information homologues representatives of the protein class LEA from different sources can be obtained and applied according to the present invention. As follows, all homologs are included, so far as they can stabilize a biological glass formed from non-reducing sugars, such as, in particular, trehalose and/or sucrose.

Exemplary representatives of the protein class LEA are presented in the following table, however these examples do not limit the present invention. The respective sequence information can easily be obtained by one of skill in the art by means of the presented data. Data bank Organism Reference (Accession No.) SWISS-PROT Gossypium hirsutum P09422 SWISS-PROT Raphanus sativus P21208 SWISS-PROT Zea mays P46517 SWISS-PROT Hordeum vulgare Q02400 SWISS-PROT Hordeum vulgare QO5191 SWISS-PROT Hordeum vulgare Q5190 SWISS-PROT Hordeum vulgare P46532 SWISS-PROT Helianthus annuus P46515 SWISS-PROT Helianthus annuus P46515 SWISS-PROT Gossypium hirsutum P09411 SWISS-PROT Gossypium hirsutum P46518 SWISS-PROT Gossypium hirsutum P09443 SWISS-PROT Gossypium hirsutum P13940 SWISS-PROT Gossypium hirsutum P09444 SWISS-PROT Gossypium hirsutum P46521 SWISS-PROT Gossypium hirsutum P4522 SWISS-PROT Brassica napus P13934 SWISS-PROT Gossypium hirsutum P13939 SWISS-PROT Zea mays Q42376 SWISS-PROT Tricitum aestivum Q03968 TrEMBL Phaseolus vulgaris O24442 TrEMBL Arabidopsis thaliana O64820 TrEMBL Arabidopsis thaliana O65148 TrEMBL Arabidopsis thaliana O80576 TrEMBL Arabidopsis thaliana O81483 TrEMBL Oryza sativa P83196 TrEMBL Oryza sativa P83197 TrEMBL Glycine max P93165 TrEMBL Glycine max Q01527 TrEMBL Tricitum aestivum Q03967 TrEMBL Arabidopsis thaliana Q39138 TrEMBL Citrus sinensis Q39466 TrEMBL Chlorella vulgaris Q39660 TrEMBL Gossypium hirsutum Q39793 TrEMBL Gossypium hirsutum Q39797 TrEMBL Glycine soja Q39919 TrEMBL Onoclea sensibilis Q40697 TrEMBL Picea glauca Q40842 TrEMBL Picea glauca Q40843 TrEMBL Picea glauca Q40848 TrEMBL Picea glauca Q40858 TrEMBL Picea glauca Q40869 TrEMBL Zea mays Q41804 TrEMBL Oryza sativa Q8S4X7 TrEMBL Brassica campestris Q8S8Z2 TrEMBL Brassica napus Q8S8Z2 TrEMBL Arabidopsis thaliana Q96273 TrEMBL Oryza sativa Q9AWZ5 TrEMBL Arabidopsis thaliana Q9FG31 TrEMBL Arabidopsis thaliana Q9FK14 TrEMBL Arabidopsis thaliana Q9FK15 TrEMBL Arabidopsis thaliana Q9FKV7 TrEMBL Oryza sativa Q9FPB2 TrEMBL Arabidopsis thaliana Q9LF88 TrEMBL Oryza sativa Q9LGL8 TrEMBL Arabidopsis thaliana Q9LT74 TrEMBL Euphorbia esula Q9M556 TrEMBL Arabidopsis thaliana Q9S7S3 TrEMBL Oncolea sensibilis Q9S2B2 TrEMBL Arabidopsis thaliana Q9SKPO TrEMBL Chlorella vulgaris Q9SLP7 TrEMBL Arabidopsis thaliana Q9XID7 TrEMBL Arabidopsis thaliana Q9ZPW6 TrEMBL Glycine max Q9ZTZ2

The LEA proteins or polypeptides proposed according to the present invention can be isolated from natural sources, produced recombinantly or synthesized. The processes to be used are well known to one of ordinary skill in the art.

A further aspect of the present invention relates to the provision of a method for stabilizing or preserving biomolecules in which the molecules to be protected are incubated in the composition according to the present invention. After a sufficient period of incubation, the preparation can then, for example, be dried at room temperature and stored until use without cooling. Insofar as the process is used for stabilization of biomolecules immobilized on particular surfaces, these loaded surfaces are covered with the composition according to the present invention. This can, for example, take place by spraying the composition on the surface or by immersing the surface in the composition. In the case of an immersion method, the surface should, preferably, be withdrawn with a speed of about 2 mm per second so that a uniform wetting with the composition according to the present invention can take place.

A further aspect of the present invention relates to the provision of surfaces that have been covered with the composition of the present invention. Biomolecules are directly or indirectly immobilized on preferred surfaces and are stabilized or preserved with the composition of the present invention. Particularly preferred surfaces are components of analytical and/or diagnostic devices, such as, for example, biochips, sensor chips, microtiter plates, test tubes, and the like. The material of the surface is not limited and can, for example be selected from glass, quartz glass, quartz, silicon, polymers (PMMA, polystyrene, polyethylene, polypropylene, PVC, etc.), and membranes such as, for example, nitrocellulose, nylon and microfiber membranes, as well as paper.

The presently used term “biological molecule” and “biomolecule” encompass any substances and compounds substantially of biological origin that have properties that are relevant within the framework of scientific, diagnostic and/or pharmaceutical applications. Encompassed are not only native molecules, such as those that can be isolated from natural sources, but also forms, fragments and derivatives derived therefrom, as well as recombinant forms and artificial molecules, as long as at least one property of the native molecules is present. Preferred biomolecules are those that can be applied for analytical, diagnostic and/or pharmaceutical purposes, such as nucleic acids and their derivatives (DNA, RNA, PNA, LNA, ribozymes, oligonucleotides, plasmids, chromosomes), peptides and proteins (enzymes, receptor proteins, protein complexes, peptide hormones, antibodies), as well as biologically active fragments thereof, carbohydrates and their derivatives such as, in particular, glycosylated proteins and glycosides, and fats, fatty acids and lipids.

It is clear to one of skill in the art that the composition and processes according to the present invention can also be applied to cellular tissues and complete cells as well as portions thereof (organelles, membranes and membrane fragments, etc.) as long as they are carriers of the above biomolecules. For this reason, tissues, cells and portions thereof are basically encompassed by the term “biomolecules”.

The used terms “stabilization” and “preservation” relate to the structural or functional integrity of biomolecules and the biological properties based thereon. The required activity of a biomolecule for a particular application requires, for example, the extensive maintenance of its primary, secondary and/or tertiary structure. The biological activity of a nucleic acid probe comprises, for example, its property for forming a hybridization complex with a nucleic acid target which is complementary to the probe. The biological activity of an antibody comprises, for example, a specific binding of its antigen.

The invention is more closely elucidated by means of the following example.

Coating of Surfaces

The formation of a composition according to the present invention results by dissolving 0.1% (weight/volume) recombinant LEA-protein and 5% (weight/volume) trehalose and 100 ml phosphate buffer (50 mM phosphate, 100 mM NaCl, pH 6.8). Subsequently, 0.02% (weight/volume) sodium azide is added as a cytotoxic agent against microbial impurities. After sterile filtration of the solution obtained using a 0.2 micrometer filter, the result is stored in an autoclaved sterile bottle.

In order to layer suitable surfaces according to the present invention, biochips (for example microscope slides) with immobilized biomolecules are immersed in the solution in a clean room (or sterile work bench) and are drawn out of the solution with a speed of 2 mm per second. In this manner a thin layer of the trehalose/LEA solution is dried on the chip and leads to the stabilization of the biomolecules.

For storage, the chips with stabilized biomolecules treated in this manner can be packaged in plastic bags and stored at room temperature under an atmosphere of nitrogen. The bags can preferably be packed in a carton sealed from light in order to prevent possible photo degradation of the proteins.

For use, the chips are taken from their packaging and rehydrated (5 minutes, RT) with assay buffer (for example PBS buffer). Next, the sample liquid is incubated directly on the chip and the assay is carried out. All kinds of receptor ligand interactions known in the art can be carried out. If desired the removal of the stabilizer can also be left out. In this case the sample is directly put onto the trehalose layered surface. This is above all practical if it has been previously shown that neither trehalose nor LEA protein interfere with the detection. 

1. A composition for stabilizing or preserving biomolecules, comprising: at least one non-reducing disaccharide; and at least one protein or polypeptide of the LEA class.
 2. A composition according to claim 1, wherein the non-reducing disaccharide is selected from the group consisting of trehalose (D-glucopyranosyl-D-glucopyranoside), sucrose (β-D-fructofuranosyl-α-D-glucopyranoside), as well as derivatives thereof.
 3. A composition according to claim 1, wherein the non-reducing disaccharide is trehalose.
 4. A composition according to claim 1, wherein the at least one protein or polypeptide of the LEA class has a motif comprising eleven amino acids, which is characterized by the following general formula (SEQ ID NO 1): (1)-(2)-(3)-(4)-(5)-(6)-(7)-(8)-(9)-(10)-E, wherein (1) signifies K or T, (2) signifies A, G, K, M or T, (3) signifies R, D, A, E, Q or K, (4) signifies E, K or S, (5) signifies T, F, Y or A, (6) signifies K, R, T or A, (7) signifies D, E or Q, (8) signifies S, R, Y or K, (9) signifies A or T, and (10) signifies G, A or R.
 5. A composition according to claim 1, wherein said at least one protein or polypeptide of the LEA class is of LEA subclass 3 and further has an amino acid sequence coded by a nucleotide sequence selected from the group consisting of those deposited in GenBank under the accession number AF423069 or S39475.
 6. A composition according to claim 1, wherein said at least one protein or polypeptide of the LEA class is of LEA subclass 3 and further has a motif comprising 11 amino acids, wherein the motif is selected from the group consisting of: (a) K-T-A-E-F-R-D-S-A-G-E, (SEQ ID NO. 2) (b) K-G-Q-E-F-K-E-R-A-G-E, (SEQ ID NO. 3) (c) K-A-E-E-T-K-Q-R-A-G-E, (SEQ ID NO. 4) (d) K-M-D-E-T-K-Q-R-A-G-E, (SEQ ID NO. 5) (e) K-A-R-K-T-K-D-S-A-A-E, (SEQ ID NO. 6) (f) K-A-K-E-Y-K-D-Y-T-A-E, (SEQ ID NO. 7) (g) K-A-R-E-T-T-E-K-A-R-E, (SEQ ID NO. 8) and (h) T-K-D-S-A-A-E-K-A-R-E. (SEQ ID NO. 9)


7. A composition according to claim 1, wherein said non-reducing disaccharide is present at from 0.01 to 15 weight percent in relation to a ready-to-use solution and said protein or polypeptide of the LEA class is present at from 0.00001 to 1 weight percent in relation to the ready-to-use solution.
 8. A process for stabilizing or preserving a biomolecule& comprising the steps of: providing a composition in accordance with claim 1; and incubating a biomolecule in the composition.
 9. A process for stabilizing or preserving a biomolecules immobilized on a surface comprising the steps of: providing a surface having a biomolecules immobilized thereon; and covering the surface with the composition as defined in claim
 1. 10. A surface with an immobilized and stabilized or preserved biomolecules, obtained by the process as defined in claim
 9. 11. A surface, covered with the composition as defined in claim
 1. 12. An analytic and/or diagnostic device, comprising a surface according to claim
 10. 13. An analytic and/or diagnostic device, comprising a surface as defined in claim
 11. 14. A device according to claim 13 selected from the group consisting of biochips, sensor chips, microtiter plates, test tubes and culture dishes.
 15. (canceled)
 16. A process for stabilizing or preserving biomolecules comprising the steps of: (a) providing a biomolecules immobilized on a surface; and (b) covering the surfaces with a composition comprising: i. at least one non-reducing disaccharide; and ii. at least one protein of polypeptide of the LEA class.
 17. A process according to claim 16, wherein the non-reducing disaccharide is selected from the group consisting of trehalose (D-glucopyranosyl-D-glucopyranoside), sucrose β-D-fructofuranosyl-α-D-glucopyranoside), and derivatives thereof.
 18. A process according to claim 16, wherein the non-reducing disaccharide is trehalose.
 19. A process according to claim 16, wherein the at least one protein or polypeptide of the LEA class has a motif comprising eleven amino acids, which is characterized by the following general formula (SEQ ID NO 1): (1)-(2)-(3)-(4)-(5)-(6)-(7)-(8)-(9)-(10)-E, wherein (1) signifies K or T, (2) signifies A, G, K, M or T, (3) signifies R, D, A, E, Q or K, (4) signifies E, K or S, (5) signifies T, F, Y or A, (6) signifies K, R, T or A, (7) signifies D, E or Q, (8) signifies S, R, Y or K, (9) signifies A or T, and (10) signifies G, A or R.
 20. A process according to claim 16, wherein said at least one protein or polypeptide of the LEA class is of LEA-subclass 3 and further has an amino acid sequence coded by a nucleotide sequence selected from the group consisting of those deposited in GenBank under the accession number AF423069 or S39475.
 21. A process according to claim 16, wherein said at least one protein or polypeptide of the LEA class has a motif comprising 11 amino acids, wherein the motif is selected from the group consisting of: (a) K-T-A-E-F-R-D-S-A-G-E, (SEQ ID NO. 2) (b) K-G-Q-E-F-K-E-R-A-G-E, (SEQ ID NO. 3) (c) K-A-E-E-T-K-Q-R-A-G-E, (SEQ ID NO. 4) (d) K-M-D-E-T-K-Q-R-A-G-E, (SEQ ID NO. 5) (e) K-A-R-K-T-K-D-S-A-A-E, (SEQ ID NO. 6) (f) K-A-K-E-Y-K-D-Y-T-A-E, (SEQ ID NO. 7) (g) K-A-R-E-T-T-E-K-A-R-E, (SEQ ID NO. 8) and (h) T-K-D-S-A-A-E-K-A-R-E. (SEQ ID NO. 9)


22. A process according to claim 16, wherein said non-reducing disaccharide is present at from 0.01 to 15 weight percent in relation to a ready-to-use solution and said protein or polypeptide of the LEA class is present at from 0.00001 to 1 weight percent in relation to the ready-to-use solution.
 23. (canceled)
 24. A process for the production of a surface with immobilized and stabilized or preserved biomolecules comprising the steps of: (a) providing a surface with a biomolecule immobilized, stabilized, or preserved thereon; and (b) covering the biomolecule* with a composition comprising: i. at least one non-reducing disaccharide; and ii. at least one protein or polypeptide of the LEA class.
 25. A surface with immobilized and stabilized, or preserved biomolecules obtained through the process as defined by claim
 16. 26. A component of an analytical and/or diagnostic device, wherein the component is a surface having a biomolecule immobilized and stabilized or preserved thereon, covered with a composition comprising: at least one non-reducing disaccharide; and at least one protein or polypeptide of the LEA class.
 27. A surface of a material selected from the group consisting of glass, quartz glass, quartz, silicon, polymers, nitrocellulose, nylon and micro fiber membranes, and paper, wherein the surface includes an immobilized and stabilized, or preserved biomolecules, thereon, covered with a composition comprising: at least one non-reducing disaccharide; and at least one protein or polypeptide of the LEA class.
 28. A surface according to claim 27, wherein said non-reducing disaccharide is present at from 0.0:1 to 15 weight percent in relation to a ready-to-use solution and said protein or polypeptide of the LEA class is present at from 0.0000 to 1 weight percent in relation to the ready-to-use solution.
 29. (canceled)
 30. A surface according to claim 27, wherein the non-reducing disaccharide is selected from the group consisting of trehalose (D-glucopyranosyl-D-glucopyranoside), sucrose (β-D-fructofuranosyl-α-D-glucopyranoside), as well as derivatives thereof.
 31. A surface according to claim 27, wherein the non-reducing disaccharide is trehalose.
 32. A surface according to claim 27, wherein the at least one protein or polypeptide of the LEA class has a motif comprising eleven amino acids, which is characterized by the following general formula (SEQ ID NO 1): (1)-(2)-(3)-(4)-(5)-(6)-(7)-(8)-(9)-(10)-E, wherein (1) signifies K or T, (2) signifies A, G, K, M or T, (3) signifies R, D, A, E, Q or K, (4) signifies E, K or S, (5) signifies T, F, Y or A, (6) signifies K, R, T or A, (7) signifies D, E or Q, (8) signifies S, R, Y or K, (9) signifies A or T, and (10) signifies G, A or R.
 33. A surface according to claim 27, wherein said at least one protein or polypeptide of the LEA class is of LEA-subclass 3 and further has an amino acid sequence coded by a nucleotide sequence selected from the group consisting of those deposited in GenBank under the accession number AF423069 or S39475.
 34. A surface according to claim 27, wherein said at least one protein or polypeptide of the LEA class is of L EA subclass 3 and further has a motif comprising 11 amino acids, wherein the motif is selected from the group consisting of: (a) K-T-A-E-F-R-D-S-A-G-E, (SEQ ID NO. 2) (b) K-G-Q-E-F-K-E-R-A-G-E, (SEQ ID NO. 3) (c) K-A-E-E-T-K-Q-R-A-G-E, (SEQ ID NO. 4) (d) K-M-D-E-T-K-Q-R-A-G-E, (SEQ ID NO. 5) (e) K-A-R-K-T-K-D-S-A-A-E, (SEQ ID NO. 6) (f) K-A-K-E-Y-K-D-Y-T-A-E, (SEQ ID NO. 7) (g) K-A-R-E-T-T-E-K-A-R-E, (SEQ ID NO. 8) and (h) T-K-D-S-A-A-E-K-A-R-E. (SEQ ID NO. 9)


35. An analytical and/or diagnostic device, comprising a surface as defined in claim
 27. 36. A device according to claim 35 selected from the group consisting of biochips, sensor chips, microtiter plates, test tubes and culture dishes.
 37. An analytic and/or diagnostic device, comprising a surface as defined in claim
 10. 